It was shown that the heavy chain and Beta-2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, Beta-2 microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, NP (85-94), crystallizes under conditions previously used to crystalize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence. HLA-A2 has also been assembled from subunits expressed separately in E. coli. A peptide that is known to be recognized by human cytotoxic T lymphocytes (CTLs) in association with HLA-A2 is a necessary component of the reconstitution mixture. The HLA-A2 heavy chain only becomes soluble in physiological solutions if both beta2m and the NP peptide are present. The reconstituted HLA-A2 complex is recognized by monoclonal antibodies that are specific for the native HLA-A2/beta2m heterodimer. The isoelectric point of the reconstituted complex depends upon which peptide is used, confirming that the peptide is a component of the reconstituted complex. To better understand the specificity of peptide binding by MHC class I molecules, the capacity of a panel of unrelated peptides to compete for the presentation of viral peptides presented by HLA-A3 and HLA-B27 was evaluated. These results were compared to study on the presentation of an influenza nucleoprotein peptide by HLA-B27 and a matrix peptide by HLA-2 to the appropriate peptide-specific CTL lines. Out of 41 peptides tested, only 5 bound to more than one of the MHC molecules analyzed. Thus, each class I molecule appears to have a functionally distinct peptide binding site as reflected by their ability to bind largely non-overlapping sets of peptides.